1. Field of the Invention
The present invention relates to a detection method for an antigen such as a chemical compound, a peptide, a protein, an RNA, a DNA, a cell (proteins released in situ), or a virus particle (proteins released in situ). In particular, the present invention provides a method and composition useful for performing ELISA assays, which also can be used for Western blot and Dot blot assays.
2. Description of the Prior Art
Immunological methods have become important tools useful for detecting antigens including, for example, peptides, proteins, nucleic acids, biological cells, and virus particles. A wide variety of methods have been developed for the detection or quantitation of antigens. Among them, Western Blot, Dot Blot, ELISA and Immunohistology are the four most commonly used methods.
Enzyme-linked Immunosorbent Assays (ELISAs), which combine the high specificity of antibodies with the high sensitivity of enzyme assays by using antibodies or antigens coupled to an easily assayed enzyme that possesses a high turnover number such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), are very useful tools both for determining antibody concentrations (antibody titer) in sera as well as for detecting the presence of antigen.
There are two main variations on this method: ELISA can be used to detect the presence of antigens that are recognized by a detection agent or it can be used to test for detection agents that recognize an antigen. There are many different types of ELISAs. Four of the most common types of ELISA are “Direct ELISA,” “Indirect ELISA,” “Sandwich ELISA” and Cell-based ELISA (C-ELISA).
A conventional direct ELISA (FIG. 1) is comprised of the following steps: (i) coating a solid phase with an antigen dissolved in a coating buffer; (ii) incubating the solid phase from Step (i) with a blocking reagent for 1 hour to block non-specific binding sites on the solid phase; (iii) washing the solid phase from Step (ii) three times with PBS or PBST for 1 min each; (iv) incubating the solid phase from Step (iii) with a primary detection agent which binds to the antigen; (v) washing the solid support from Step (iii) five times for 1 min each in PBS or PBST to remove the non-specifically bound primary detection agent; and (vi) using a detection system such as UV, fluorescence, chemiluminescence or other detection methods to detect the bound primary detection agent. The primary detection agent can be, without limitation, a detection agent linked (coupled) to a fluorescent dye, or a reporter enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), which can convert a colorless substrate to a colored product whose optical densities can be measured on an ELISA plate reader at target wavelengths.
A conventional indirect ELISA (FIG. 3) is comprised of the following steps: (i) coating a solid phase with an antigen dissolved in a coating buffer; (ii) incubating the solid phase from Step (i) with a blocking reagent for 1 hour to block non-specific binding sites on the solid phase; (iii) washing the solid phase from Step (ii) three times with PBS or PBST for 1 min each; (iv) incubating the solid phase from Step (iii) with a primary detection agent diluted in a solution for 1 hour; (v) washing the solid support from Step (iv) three times for 1 min in PBS or PBST to remove the non-specifically bound primary detection agent; (vi) incubating the solid support from step (v) with a secondary detection agent diluted in a solution for 1 hour; (vii) washing the solid support from Step (vi) five times for 1 min each in PBS or PBST to remove the non-specifically bound secondary detection agent; and (viii) using a detection system such as UV, fluorescence, chemiluminescence or other methods to detect the bound secondary detection agent. The secondary detection agent binds the primary detection agent. The secondary detection agent can be, without limitation, a detection agent linked (coupled) to a reporter enzyme such as alkaline phosphatase (AP) or horseradish peroxidase (HRP), which can convert a colorless substrate to a colored product whose optical densities can be measured on an ELISA plate reader at target wavelengths.
The complete direct ELISA procedure involves at least three incubation steps: the first is incubation between the solid support and the antigen; the second is incubation between the solid support and the blocking reagent; and the third one is incubation between the solid support and the primary detection agent. The incubation step is a two-phase reaction and involves the binding reaction between the antigen on the solid support and the detection agent.
The complete indirect ELISA procedure involves at least four incubation steps: the first is incubation between the solid support and an antigen; the second is incubation between the solid support and the blocking reagent; the third one is incubation between the solid support and the primary detection agent; and the fourth is incubation between the solid support and the secondary detection agent. The incubation step is a two-phase reaction and involves the binding reaction between the antigen on the solid support and the detection agent.
In a conventional direct ELISA, the first incubation step, antigen coating, takes at least 2 hours and each other incubation step takes about 1 hour. A conventional direct ELISA, as described above, therefore, takes at least 4 hours.
In a conventional indirect ELISA, the first incubation step, antigen coating, takes at least 2 hours and each other incubation step takes about 1 hour. A conventional indirect ELISA, as described above, will take at least 5 hours. Because conventional direct and indirect ELISA consumes valuable time, there is a need for a simple and rapid process to address these conventional time-consuming assays.
The cell-based ELISA (C-ELISA) is a moderate throughput format for detecting and quantifying cellular proteins including post-translational modifications associated with cell activation (e.g., phosphorylation and degradation). Cells are plated, treated according to experimental requirements, fixed directly in the wells, and then permealized. After permealizing, fixed cells are treated similar to a conventional immunoblot, including blocking, incubation with a first antibody, washing, incubation with a second antibody, addition of chemilumescent substrates and development.
In 1971, Engvall and Perlmann (Immunochem., 8:871-874, 1971) coined the term “enzyme-linked immunosorbent assay,” which is better known by the acronym “ELISA”, to describe an enzyme-based immunoassay method which is very useful for measuring antigen concentrations. Since then, ELISA has not only become one of the most commonly used methods for protein and antibody detection and identification but also the basic immunoassay upon which many of the modern assays are based.
A rapid method for microwave mediated enzyme-linked immunosorbent assay (M-ELISA) (U.S. Pat. No. 6,498,016) was developed to perform ELISA rapidly. However, this procedure requires a carefully-controlled microwave which needs optimization.
Although there have been substantive improvements in all of these immuno-detection methods, including the quality of reagents, solid phase plates and plastics, microplate readers, washers, and statistical software, the basic methodology has remained virtually unchanged.